The need to preserve sperm samples, intended for artificial insemination (AI), through freezing for periods of time adequate to test donors for HIV has focussed attention on the damage to the sperm cells brought about by the process of cryopreservation. The percentage of motile cells is decreased and there appears to be lowered pregnancy rates in AI programs, attributed to loss of fertilizing ability of cryopreserved sperm. The cause of damage is unknown. The underlying hypothesis of this proposal is that the freeze-thaw process involved in cryopreservation renders sperm highly sensitive to damage through peroxidation of membrane phospholipids. The objective of this proposal is to test this hypothesis through three specific aims, 1) to quantitate phospholipid loss, lipid peroxidation rate, and inactivation of the protectant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPX) in human sperm samples in the fresh state and after freeze-thaw, under aerobic and anerobic conditions, and in the presence and absence of peroxidation protectants. 2) To determine the percentages of sperm in a given sample which undergo the physiological, zona-induced acrosome reaction and the non-physiological reaction of acrosomal loss with concomitant loss of the equatorial segment, in samples in the fresh state and after freeze-thaw. Correlation will be sought with the degree of peroxidative phospholipid loss under the different conditions. Since sperm in the first category can fertilize eggs while those in the second category cannot, this determination should provide a link between peroxidative damage brought about by cryopreservation, and fertilizing ability 3) To compare the rate of incorporation of exogenous fatty acids into sperm recovered from crypreservation and those in the fresh state, and to utilize this incorporation procedure to make up for peroxidative phospholipid loss as a means of repairing cryodamage. The thin layer chromatography (TLC) methods for phospholips and assays for enzymes acting as protectants against peroxidation, which have been developed during extensive studies of lipid peroxidation in mammalian sperm in these laboratories, will be applied to this study. The results of the proposed research are expected to form a completely new approach to the development of cryopreservation procedures for human sperm.